Embryonic stem cells (ESCs) were first derived from mice and are now available from a variety of mammalian systems, including human. They are characterized by nearly unlimited self-renewal in an undifferentiated state under defined culture conditions while retaining differentiation capacity (Evans et. al., Nature, 292:154-156, 1981; Martin, Proc. Natl. Acad. Sci. U. S. A. 78: 7634-7638, 1981; Smith, Cold Spring Harbor Laboratory Press, New York, 2001). During differentiation in vitro, embryonic stem cells are able to develop into various kinds of specialized somatic cell types and recapitulate processes of early embryonic development. Thus, embryonic stem cells hold promise as an unlimited source for various clinical and biotechnological applications (Brustle, Science, 285:754-756, 1999; Martin, Proc. Natl. Acad. Sci. U. S. A. 78: 7634-7638, 1981; Li et al., Curr. Biol., 8:971-974, 1998; Pera et al., J. Cell. Sci., 113:5-10, 2000).
Currently a few molecular regulators are known to participate in the self-renewal and pluripotency of mouse embryonic stem (mES) cells. A POU family transcription factor Oct4, the classical marker of all pluripotent cells, is specifically expressed in pre-implantation embryos, epiblast, germ cells and pluripotent stem cell lines including embryonic stem cells, embryonic germ (EG) cells and embryonic carcinoma (EC) cells (Palmieri et al., Dev. Biol., 166:259-267, 1994; Yeom et al., Development, 122:881-894, 1996). Oct4 plays a critical role in the establishment and maintenance of pluripotent cells in a pluripotent state (Nichols et al., Cell, 95:379-391, 1998; Niwa et al., Nat. Genet., 24:372-376, 2000; Pesce et al., BioEssays, 20:722-732, 1998). Leukemia inhibitory factor (LIF) can maintain self-renewal of mouse embryonic stem cells through activation of Stat3. Oct4 and Stat3 each interact with various cofactors and regulate the expression of multiple target genes (Niwa et al., Gene Dev., 12:2048-2060, 1998). Two other transcription factors, Sox2 and FoxD3, have been shown to be essential for pluripotency in mice embryos (Avilion et al., Gene Dev., 17:126-140, 2003; Hanna et al., Gene Dev., 16:2650-2661, 2002). More recently, it was found that the homeoprotein Nanog is capable of maintaining self-renewal of mouse embryonic stem cell, independently of LIF/Stat3 (Chambers et al., Cell, 113:643-655, 2003; Mitsui et al., Cell, 113:631-642, 2003).
The first human embryonic stem cell line was established only recently (Thomson et al., Science, 282:1145-1147, 1998) and 12 lines are publicly available worldwide (NIH Human Embryonic Stem Cell Registry). Despite their great potential, human embryonic stem cells have not been a prolific source of information. This is mainly due to the technical difficulties in cell culture. Maintaining and expanding human embryonic stem cells require laborious and skill-intensive procedures. Moreover, the population-doubling time of human embryonic stem cells is almost three times longer than that of mouse embryonic stem cells (Amit et al., Dev. Biol., 227:27 1-278, 2000). There exist apparent differences in the characteristics of human embryonic stem cells compared to mouse embryonic stem cells in many aspects, including the regulation of self-renewal. Of the regulators found in mice, only a few including Oct4 play similar regulatory roles in human embryonic stem cells. Others such as LIF do not affect human embryonic stem cells in maintaining their self-renewal (Reubinoff et al., Nat. Biotechnol., 18:399-404, 2000). Dissecting the regulatory mechanism in human embryonic stem cells will greatly enhance the understanding of stem cells as well as their application.
Recent advances in small RNA research have implicated microRNAs (hereinafter, referred to as ‘miRNAs’) as important regulators of development and differentiation. miRNAs constitute a large family of non-coding small RNAs of ˜22 nucleotides (nt) in length. Our understanding of miRNA function originates from studies of the developmentally regulated miRNAs lin-4 (Olsen and Ambros, Dev. Biol., 216:671-680, 1999; Lee et al., Cell, 75: 843-854, 1993; and Wightman et al., Cell, 75:855-862, 1993) and let-7 (Reinhart et al, Nature, 403:901-906, 2000) in Caenorhabditis elegans. By binding and inhibiting the translation of the target mRNA, the lin-4 and let-7 RNAs play an important role in regulating the timing of larval development. Another example is bantam RNA from Drosophila melanogaster, which is expressed in a temporal and tissue-specific manner during development, suppressing apoptosis and stimulating cell proliferation by inhibiting translation of hid mRNA (Brennecke et al., Cell, 113:25-26, 2003). Several mouse miRNAs including miR-181 were shown to modulate hematopoiesis (Chen et al., Science, 303:83-86, 2003). In plants, miRNAs show a high degree of complementarity to transcription factors that are significant in development (Aukerman and Sakai, Plant Cell, 2003; Chen, Science, 2003; Llave et al., Science, 297:2053-2056, 2002b; Palatnik et al., Nature, 425:257-263, 2003 and Rhoades et al., Cell, 110:513-520, 2002). These miRNAs induce target mRNA cleavage or translational repression, thereby facilitating plant development and organogenesis.
The expression of miRNAs is often regulated in tissue-specific and developmental stage-specific manners (Aravin et al., Dev. Cell, 5:337-350, 2003; Krichevsky et al., RNA, 9:1274-1281, 2003; Lagos-Quintana et al, Science, 294:853-858, 2002; Pasquinelli et al., Nature, 408-86-89, 2000 and Sempere et al., Dev. Biol. 259:9-18, 2003), although the regulatory mechanism is still largely unknown. The present inventors have previously shown that miRNAs are transcribed as long primary transcripts (termed pri-miRNAs) (Lee et al., EMBO J., 21:4663-4670, 2002). These primary transcripts are first trimmed into approximately 70 nt stem-loop forms (called pre-miRNAs) by the RNase III type protein, Drosha, in the nucleus (Lee et al., Nature, 425:415-419, 2003). Following this initial processing, pre-miRNAs get exported to the cytoplasm by Exportin-5 (Lund et al., Science, 303:95-98, 2003 and Yi et al., Genes Dev., 2003) and are subject to a second processing to generate the final product of approximately 22 nt mature miRNAs, by another RNase III type protein Dicer (Grishok et al., Cell, 106:23-24, 2001; Hutvagner et al., Science, 293:834-838, 2001; Ketting et al., Genes Dev., 15:2654-2659, 2001; and Knight and Bass, Science, 293:2269-2271, 2001). This stepwise processing and compartmentalization may allow for the fine regulation of miRNA biogenesis at multiple steps.
More than 300 miRNAs have been reported in diverse eukaryotic organisms so far (Aravin et al., Dev. Cell, 5:337-350, 2003; Dostie et al, RNA, 9:180-186, 2003; Grad et al., Mol. Cell., 11:1253-1263, 2003; Lagos-Quintana et al., Science, 294:853-858, 2001; Lagos-Quintana et al., Curr Biol. 12:735-739, 2002; Lagos-Quintana et al., RNA, 9:175-179, 2003; Lai et al., Genome Biol., 4, R42, 2003; Lau et al., Science, 294:858-862, 2001; Lee and Ambros, Science, 294:862-864, 2001; Lee et al., Cell, 75:843-854, 1993; Lim et al., Genes Dev., 2, 2, 2003b; Llave et al., Plant Cell, 14:1605-1619, 2002a; Mourelatos et al., Genes Dev., 16:720-728, 2002; Park et al., Curr. Biol., 12:1484-1495, 2002; Reinhart et al., Nature, 403-901-906, 2000 and Reinhart et al., Genes Dev., 16:1616-1626, 2002). The majority of miRNA genes were discovered through cDNA cloning from size-fractionated RNA samples. Recently, additional miRNA genes have been identified using computational procedures from the vertebrates, C. elegans and Drosophila. A bioinformatic study suggested that there exist 200-255 miRNAs in humans, accounting for almost 1% of the predicted genes (Lim et al., Science, 299, 1540, 2003a). If the prediction is correct, about 100 miRNA genes remain to be identified in humans because 152 miRNAs have been reported, of which 109 miRNAs have been experimentally validated (Brennecke and Cohen, Genome Biol., 4, 228, 2003). miRNAs that are expressed only in specific developmental stages or conditions would be difficult to be cloned or validated. However, miRNAs have not been isolated yet from human embryonic stem cells.